Leukemia cell lines MV4-11 (MLL-AF4), MOLM13 (MLL-AF9), K562 (BCR- ABL), REH (ETV6-RUNX1) and MOLT4 (T lymphoblast) were obtained from the American Type Culture Collection (ATCC) and KOPN 8 (MLL-ENL) cells were obtained from the Leibniz Institute DSMZ. These cells were cultured in RPMI 1640
hFMR2: LWVKIDLDLLSRV hAF4: LMVKITLDLLSRI PFWT (Penetratin)-LWVKIDLDLLSRV PFmut (Penetratin)-LWEKSDLDLLSRV Penetratin RQIKIWFQNRRMKWKK SPK111- modified Tat(dL)(Nle)VOrnIDL(dD)L(dL)-CONH2 SPK110 –modified Tat(dL)(Nle)VDIDL(dD)L(dL)-CONH2
Modified Tat - (dR)(dK)(dK)(dR)(dR)Orn(dR)(dR)(dR)(βA)
Figure 8 Schematic of peptide design based on AF9 interacting domain of AF4 The amino acid sequence of AF9 binding domain of human AF4 and FMR2 is shown in figure. The peptide FMR wild type (PFWT) sequence is based on FMR2 amino acids. The mutant version of the AF4 mimetic peptide PFmut has mutated amino acids which are in red. The amino acid sequence of the protein transduction domain Penetratin has also been shown. The peptide SPK111 and SPK110 generated by stereomeric and isomeric substitution of amino acids is also shown. Substituted dextro stereoisomers of amino acids are represented by (d), and the beta isomers of an amino acid are represented by (β). Some of the lysine residues are substituted with ornithine represented by Orn and tryptophan is replaced with Norleucine represented by (Nle). Ornithine residue (Orn) in SPK111 forms a salt bridge interaction with AF9. In SPK110, Orn is substituted with negatively charged aspartic acid D.
medium (ATCC, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA), 1.1 % penicillin/streptomycin (Pen/Strep) (Invitrogen), and 2.2% glutamine ( Cell Grow, Mediatech, Manassas, VA).
HEK293T cells (Clontech) and HeLa (ATCC) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Cellgrow, Mediatech, Manassas, VA), supplemented with 10% FBS, and 1.1% Pen/Strep. All cells were incubated at 37oC with 5% carbon dioxide.
Peptide Stock
The AF4 mimetic peptide, SPK111 (modified Tat- (dL)(Nle)VOrnIDL(dD)L(dL) or its mutant control SPK110 (modified Tat-(dL)(Nle)VDIDL(dD)L(dL) were synthesized and purified by HPLC to > 85% purityby New England Peptide (Gardner, MA). The modified Tat protein transduction sequence is ((dR)(dK)(dK)(dR)(dR)- O(dR)(dR)(dR)(bA)). A stock of 37.5mg/ml of SPK111 or SPK110 was dissolved in cell culture grade 99% pure Dimethyl sulfoxide (DMSO) (Edward lifesciences, CA) to obtain a 1000x stock. Aliquots of the stock were stored at -800C.
Cell Viability Assay
The Promega Cell Titer-Glo luminescent cell viability assay measures the total cellular ATP and can be used to quantify metabolically viable cells. This assay was used to measure the cell viability. MV4-11, MOLM13, KOPN8, K562, REH and MOLT4 were seeded at a density of 1 million cells per ml. They were exposed to SPK111 or SPK110 at concentrations of 12.5µg/ml, 25µg/ml, and 37.5µg/ml. As an additional
control, cells were exposed to the same final volume of DMSO as used in peptide treatments. Cells (100µl) were dispensed in 96 well plates in quadruplicates for each given concentration. The plates were incubated at 37οC for 24 hours. After equilibration, 100 µL of Cell Titer Glow Reagent (Promega) was added to each well and placed on a rotating platform for 2 minutes to aid cell lysis. Cells were incubated for additional 10 minutes at room temperature to stabilize the luminescent signal. Luminescence was recorded using POLAR Star Omega plate reader (BMG Labtech).
Viability of the peptide treated cells was calculated as percentage of DMSO treated cells.
Generating mouse xenografts
Six week old female NOD/SCID mice were sublethally irradiated with 250 cGy of total body irradiation. On the same day, post irradiation 2x106 MOLM13, KOPN8, MV4-11, or K562 leukemia cells were injected into the tail vein. The number of mice used in each experiment is indicated in the results section.
A post mortem analysis was performed to confirm the presence of xenografted leukemic cells at the end of each experiment. Bone marrow samples were obtained by flushing femurs with PBS. The samples were lysed in 1X RBC lysis buffer (8.3g/l ammonium chloride in 0.01M Tris-HCl pH 7.5) for 2-3 minutes. The lysed sample was centrifuged. The cell pellets were blocked with 2% FBS in PBS and stained with anti- human CD45+ FITC (BD Pharmingen # 55482) and analyzed by flow BD canto II flow cytometer and Flow Jo 2.0 software.
In vivo treatment with SPK111
In order to calculate the dose for animals we assumed that the density of mouse tissue is equivalent to that of water. Hence the in vivo equivalent of 25µg/ml will be 25mg/kg.
Treatment was started one week after the injection of leukemia cells unless otherwise indicated. Mice were injected subcutaneously with the specified dose of SPK111 or with vehicle alone (2% DMSO in PBS). Survival was measured as the time from leukemia cell injection until a moribund state developed. Extreme lethargy or paralyses were considered end points. Survival benefit was assessed by Kaplan–Meyer analysis using Prism Graph Pad software. In order to detect a significant change (p< 0.05) in mean survival from 21+/-4 days to 28+/-4 days with a power of 0.80, seven animals were needed per group (2 sample t-test, calculations performed by Dr. Rong Guo, institutional biostatistician).
Effect of SPK111 on leukemia-initiating cells/ex vivo purging of leukemic cells
2 x 106 MOLM 13 cells were incubated ex vivo with 37.5 µg/ml SPK111 or 2% DMSO in PBS for 24 hours. The cells were then collected by centrifugation at 1200 rpm for 4 mins, re-suspended in PBS and injected into sub lethally irradiated NOD/SCID mice via tail vein. Mice were monitored for 2 months after cells were injected.
Bioluminescent monitoring of leukemic growth in vivo
Leukemic progression was monitored in mice xenografted with MV4-11 cells expressing luciferase. MV4-11 cells expressing luciferase were provided to us by Dr. Scott Armstrong’s Laboratory (Dana-Farber Cancer Institute, Boston, MA). Images were acquired using the xenogen imager 10 minutes following intra peritoneal injection of 125 mg/kg of D-luciferin. Luminescent images were analyzed using the caliper life science live image software.
Hematologic Toxicity
SPK111 (50 mg/kg) or 2% DMSO in PBS was administered subcutaneously to 6- week old female C57BL/6 mice daily for 5 days. Three mice were used per group. Seven days after the administration of the last dose, mice were euthanized and whole blood was collected by heart puncture. Whole blood analysis was performed with a Hemavet 950F instrument (Drew Scientific) in order to determine any short term hematological toxicity. To determine the effect on myeloid cells, bone marrows were flushed from the femur and red blood cells were lysed in buffered ammonium chloride. Cells were then collected by centrifugation. The cells were incubated in 2% FBS in PBS for 30 minutes, followed by labeling with the following eBioscience antibodies: anti-mouse CD41 (#12- 0411-81), anti-mouse Ter119 (#17-5921-81), anti-mouse CD11b (#48-0112-80) and anti- mouse LyGr-1 (#85-5831-81). After incubation, the cells were washed twice with 2% FBS in PBS and suspended in the same buffer. Cells were analyzed using a BD Canto II flow cytometer and Flow Jo 2.0 software.
Enzyme Linked Immunosorbent assay (ELISA)
ELISA was used to measure the peptide concentration in the serum following subcutaneous injections. The anti-SPK111 antibody used in this assay is a polyclonal antibody purified from the serum of a rabbit immunized with SPK111 peptide plus an adjuvant (New England Peptides, Gardner, MA). In order to establish a standard curve for the ELISA, dilutions of SPK111 stock were prepared in FBS containing 0.05% tween-20. Standards and blank (100µl) were added to wells in triplicate. To assess the concentration of peptide in the serum, 6 week old female C57BL/6 mice were subcutaneously injected with 50mg/kg of SPK111 and were sacrificed 1 h or 3 h after treatment. Whole blood was collected by heart puncture. The whole blood was allowed to clot at room temperature and then centrifuged at 10, 000 x g at 4οC for 15 minutes. The serum was collected and 100µl loaded into the wells of a protein binding 96-well plate. (About 100-150µl of serum is obtained from a single heart puncture sample, hence it is not possible to monitor the peptide levels in the same mouse). Hence each sample well represents a single animal. Plates were covered with thin plastic film and incubated in the refrigerator at 4οC overnight. The next day the wells were washed 3 times with PBS and then incubated with 100µl of anti-SPK111 antibody diluted 1:1000 in PBST (PBS+ 0.05% Tween) for 1 hour at room temperature (RT). Wells were then washed 5 times with PBST. Horse radish peroxidase conjugated anti-rabbit IgG antibody (Invitrogen) was diluted 1:7500 in PBST and added to the wells, and incubated at room temperature for 30 minutes followed by 5 washes with PBST. The HRP activity was detected using tetramethylbenzidine (TMB)/peroxide (100µL) for 10-15 mins. The reaction was stopped
by addition of 50µl of HCl to the wells (R&D Systems, Cat. #DY999). Absorbance was measured at 450nm using a POLAR Star Omega plate reader (BMG Labtech).
Results
SPK111 is toxic to leukemia cells
Incubation of leukemic cells with the AF4 mimetic peptide, PFWT, results in decreased viability. The same decrease is not observed on incubation of the peptide with peripheral blood derived mononuclear cells (Palermo et al., 2008; Srinivasan et al., 2004a). In order to confirm that the modified AF4 mimetic peptide, SPK111, has the same effect, viability assays were performed. The human leukemia cell lines MV4-11, MOLM13, and KOPN8 carrying MLL-AF4, MLL-AF9 and MLL-ENL translocations, respectively, were treated with 12.5µg/ml, 25µg/ml and 37.5µg/ml of the active peptide SPK111 and the mutant peptide SPK110 for 24 h. Viability after treatment was determined by quantification of cellular ATP using the Promega Cell Titer-Glow luminescent cell viability assay. Treatment with the vehicle, DMSO, served as negative control. As shown in Figure 9, the viability of MLL leukemia cell line decreases with increasing concentrations of SPK111. However, treatment with the mutant peptide, SPK110, which has a single amino acid mutation in the AF9 binding region, does not affect the viability at the same concentrations. This suggests that inhibition of the AF4- AF9 interaction results in decreased viability.
The myeloid leukemia cell line K562 expressing a BCR-ABL fusion gene, a pro- B-leukemia cell line REH expressing the ETV6-RUNX1 fusion gene, and a T-
lymphoblatic cell line MOLT4 carrying a p53 mutation were representative of non-MLL- rearranged leukemias. These leukemia cell lines lack MLL fusion genes and are comparatively less sensitive to SPK111 as shown in Figure 9.
SPK111 is ineffective against xenografted MLL leukemias
In order to determine if SPK111 treatment improves the survival of mice xenografted with human MLL leukemic cell lines, we injected mice with 2x106 MOLM13 and KOPN8 cells. The presence of the xenograft was confirmed at the end of each experiment by detecting the presence of hCD45 antigen from the extracted bone marrow derived whole blood samples (data not shown). In two independent experiments, xenografted mice were subcutaneously injected with 37.5mg/kg SPK111 or DMSO vehicle solution for 5 consecutive days. The first dose was administered 7 days after transplant. Assuming a volume of distribution of one, 37.5 mg/kg corresponds to an in
vitro dose that results in less than 15% viability of MOLM13 and KOPN8 cells.
The results depicted in Figure 10 show a trend towards improved survival. However, no statistical significance was obtained. In addition, mice in the SPK111 treatment group developed skin irritation and ulceration at the site of injections indicating toxicity to dermal tissue. Two days after tail vein injection of leukemic cells in mice, the leukemic burden is likely to be low. In the next experiment, we sought to determine if dosing when the disease burden is low improves survival. KOPN8 xenografted mice were subcutaneously injected with 37.5 mg/kg SPK111or DMSO two days after transplant. A total of five doses were administered to KOPN8 xenografted mice in
Figure 9 Treatment of leukemic cells with SPK111 results in decreased viability MV4-11, MOLM13, KOPN8, K562, REH, and MOLT4 leukemic cells were treated with increasing concentrations of SPK111 or the mutant peptide SPK110 for 24 hours. Viability was measured using Promega Cell titer-glow luminescent viability assay. Viability is expressed as a percentage of DMSO treated cells. The error bars represent % standard error. Significance is determined using student’s t-test. ‘*’ indicates p<0.01. No significant difference is indicated by n.s. IC50 calculated using ED50 plus software.
this experiment. Survival analysis shown in Figure 11 indicates that there was no survival advantage for the treatment group.
The adverse effects of SPK111 on the skin of the mice raised the possibility of secondary events like bleeding and infection. In order to minimize the effect on skin, we tested treatment with a smaller dose of SPK111 (25mg/kg) but with an increased number of doses. Dosing frequency was maintained at one dose given per day.The total amount of drug administered to KOPN8 xenografted mice during therapy was 5 mg of SPK111 over 10 doses which is 30% higher than the 3.75mg administered at a larger dose for 5 days previously. Similarly, the total amount of drug administered to MOLM13 xenografted mice during therapy was 6 mg of SPK111 over 12 doses which is 60% higher than the 3.75mg administered at a larger dose for 5 days. However, this dosing scheme had no effect on the skin lesions or the survival of MOLM 13 or KOPN8 xenografted mice (Figure 12).
Effect of SPK111 on normal hematopoiesis
It is important to determine the effects of an experimental drug on normal hematopoiesis in order to identify any acute toxicity. C57BL/6 mice were treated with a daily subcutaneous dose of 50mg/kg of SPK111, SPK110 or vehicle control for 5 consecutive days. The dose 50mg/kg is the largest amount of dose administered in any survival experiment. An analysis of the whole blood collected by heart puncture a week after dosing shows no significant differences in the total white blood cell count, platelet count or the hemoglobin level (Figure 13).
Figure 10 Survival of mice with MLL leukemia xenografts after treatment with 37.5mg/kg of SPK111 for 5 daily doses
MOLM13 (A) and KOPN8 (B) xenografted mice were injected subcutaneously with 37.5mg/kg SPK111 or 2% DMSO in PBS daily for 5 consecutive doses. The first dose was administered on day 7 after transplant. N represents the number of animals in each group and p value was calculated using log rank test (prism graph pad software). Median survival of DMSO and SPK111 treated mice is shown.
SPK111 [37.5mg/kg] / DMSO MOLM13 N=4 P=0.0849 Median survival DMSO - 19days SPK111-20.5days A B N=5 P=0.052 Median survival DMSO - 23days SPK111-26days SPK111 [37.5mg/kg] / DMSO
Figure 11 Survival of mice with MLL leukemia xenografts treated 2 days after transplant
KOPN8 xenografted mice were injected subcutaneously with 37.5mg/kg SPK111 or 2% DMSO in PBS daily for 5 consecutive doses. The first dose was administered on day 2 after transplant. N represents the number of animals in each group and p value was calculated using log rank test (prism graph pad software). Median survival of DMSO and SPK111 treated mice is shown.
N=7 P=0.6538 Median survival DMSO - 27days SPK111- 29days KOPN8 SPK111 [37.5mg/kg] / DMSO
Figure 12 The effects of frequent treatment with 25mg/kg of SPK111 on mice with MLL leukemia xenografts
MOLM13 (A) and KOPN8 (B) xenografts were injected subcutaneously with 25mg/kg SPK111 or 2% DMSO in PBS for the indicated number of doses. The first dose was administered on day 2 and day 7 after transplant for MOLM13 and KOPN8 respectively. N represents the number of animals in each group and p value was calculated using log rank test (prism graph pad software). Median survival of DMSO and SPK111 treated mice is shown. N=8 P=0.7278 12 doses administered Median survival DMSO -23 days SPK111-22 days N=5 P=0.1769 10 doses administered Median survival DMSO -28 days SPK111-30 days MOLM 13 KOPN8 A B SPK111 [25mg/kg]/ DMSO SPK111 [25mg/kg]/ DMSO
AF9 positively regulates genes that promote erythroid-megakaryoctic lineage precursor development (Pina et al., 2008). Hence, flow cytometry analysis using markers of erythroid-myeloid precursor cells was done on bone marrow samples derived from the same mice as Figure 13. We observed a two fold increase in Gr-1 expressing granulocyte macrophage precursors in samples derived from mice treated with active and mutant peptides compared to mice treated with the vehicle, suggesting that the peptide may affect the myeloid population (Figure14). Ter119 antibody binds erythroid cells from pro-
erythroblast through mature erythrocyte stages.Hypotonic lysis buffer is used to lyse the mature erythrocytes in the whole blood collected from the bone marrow of mice. Variation in the number of cells of the erythroid lineage determined using TER-119 antibody could be an effect of increased lysis of the mature erythrocytes (Figure14). CD41 is a marker of megakaryocytes and is also present on certain hematopoietic stem cell populations. CD41 labeling shows variation in the sample derived from mice treated with SPK111, however this variation is not statistically significant (Figure 14).
SPK111 can be used for purging of leukemia initiating cells
Leukemia initiating cells are slow dividing cells that evade conventional chemotherapeutics and play a role in re-establishing leukemia after a period of latency (Clevers, 2011). In order to test the effect of SPK111 on leukemia initiating cells, we treated 2X106 MOLM13 cells with 37.5µg/ml of SPK111 for 24 hrs. The viability of MOLM13 cells at this concentration in vitro is 15% or less. In order to determine if the remaining viable cells are resistant leukemic cells, the entire cell suspension was injected
Figure 13 SPK111 does not affect the whole blood composition
50mg/kg of SPK111, 50mg/kg of SPK110 or DMSO was administered to C57BL/6 mice daily for five consecutive days. Seven days after the last injection whole blood collected by heart puncture was analyzed on a hemavet. No significant differences were found in the platelet, hemoglobin and the total white blood cell count between samples derived from mice treated with vehicle, SPK111 or SPK110. Hemoglobin content in grams/deciliter (g/dL) and cell count is thousand cells per micro liter (K/µl). Each group consisted of three mice. Error bars represent standard deviation and significance difference between DMSO treatment derived sample and peptide treatment derived samples was determined using students t-test.
n.s. n.s.
n.s. n.s.
Figure 14 Effect of SPK111 on myeloid differentiation
50mg/kg of SPK111 was administered to C57BL/6 mice daily for five consecutive days. Seven days after the last injection the bone marrow was collected and labeled with fluorescent antibodies to the surface markers Gr1(Granulocyte Myeloid precursor), CD 41(Multipotent hematopoietic precursor and megakaryocyte), and Ter119 (pro- erythroblasts) and analyzed by flow cytometer. Gating was restricted to viable cells. There were three mice in each group. Error bars represent standard deviation and significance was determined using students t-test.
into the tail veins of sub-lethally irradiated NOD/SCID mice. Cells exposed to vehicle control caused fatal leukemia in all mice approximately 21 days following injection. In contrast, cells exposed ex vivo to SPK111 did not cause measurable disease in five of six animals during the 60 days of observation (Figure 15).
Similarly, 2X106 MV4-11 cells that express luciferase were cultured for 24 h in the presence of 37.5µg/ml SPK111 or vehicle control. The entire cell suspension was then injected into the tail veins of sub-lethally irradiated NSG mice. Luminescence at various time points is depicted and shows that the treated cells fail to establish leukemia (Figure 16).
Establishment of ELISA to determine serum SPK111 concentration